We plan to continue work in four closely related areas: (1) chitin synthesis in yeast; (2) chitin synthesis and glycosylation in multicellular organisms: (3) the enzymology and biology of the hyaluronan/chitin oligosaccharide synthesizing enzyme DG42, and (4) properties of chitin deacetylases and "antifungal" chitinases. Yeast chitin synthase 3 (Chs3p) is targeted to the bud-neck region of the cell where it synthesizes the chitin ring. It also functions throughout the plasma membrane to "reinforce" the cell wall when it has been weakened. Our immediate goals will be to compare targeting and activation of Chs3p when it is engaged either in building the bud-neck structure or depositing chitin in the lateral wall. In insects, chitin is present in the cuticle and in the intestinal peritrophic membrane. Insect genomes examined to date have two chitin synthase genes, while most filamentous fungi have at least six. We will study the enzymology of individual proteins that have been expressed in appropriate heterologous systems. We will also investigate when and where the genes are transcribed and where within cells the enzymes are localized. From our work as well as work in other laboratories, it is clear that some hyaluronan synthases (HAS enzymes, including DG42) are able to synthesize either hyaluronan (a polymer of alternating glucuronic acid and acetylglucosamine residues) or chitin oligosaccharide (containing only acetylglucosamine) depending on conformation of the enzyme and/or incubation conditions. In other developments, the Spaink group have shown clearly that the chitin tetrasaccharide, and only the tetrasaccharide, is able to restore development of the anterior-posterior axis in zebrafish inhibited with DG42 antisense RNA. We will continue our own enzymatic work and will initiate collaborative X-ray crystallographic and biological studies. In our fourth project, we will focus on chitin deacetylases, apparently soluble proteins that must act on nascent chitin chains before they associate into fibers. and on "antifungal" chitinases as potential drugs.